Putrescine Oxidase Vida Espinosa & Kathy WilsonBIOL 473 - Fall 2015

Background• Putrescine Oxidase is also known as PuOx• Belongs to the MAO family of flavin-containing amine oxidases1

• MAO: structural family, monoamine oxidase• Contains one flavin adenine dinucleotide (FAD) per dimer

• FAD is a cofactor used in the catalysis of PuOx • Catalyzes oxidation of many different compounds, including

putrescine• Reducing the oxygen to hydrogen peroxide concurrent with the

previous stated oxidation1

• No coenzyme is needed for reaction, due to the electron acceptor being molecular oxygen

Structure and Mechanism• Putrescine Oxidase exists as is a homodimer• Molecular Weight as homodimer: 198 kDa

• putrescine + O2 + H2O = 4-aminobutanal + NH3 + H2O2

Protein Tags • GFP tag:

• Known as Green Fluorescent Protein• Emits a green fluorescent glow

• Allows for visualization of where and/or when the protein is expressed2

• Molecular weight: 27 kDa• MBP tag:

• Known as Maltose Binding Protein • New England Biolabs developed MBP in the 1980’s3

• Promotes stability of protein• Heightens solubility within protein • Molecular weight: 42 kDa

Work Flow

Transformation of GFP-PuOx• Transformation completed using T7

Express lysY Competent E. coli• Plasmid for the expression of GFP-PuOx

obtained from University of Kansas• Plasmid included GFP-PuOx gene• Also contained gene for ampicillin resistance

• Heat shock used to disrupt cell wall to allow introduction of the plasmid DNA

• After transformation, the cells were then streaked onto an ampicillin/LB agar plate, allowing only growth of the transformed cells.

• Protein growth completed in 1X LB Broth, incubated overnight• Over-expression was completed after 3 hours and 30 minutes

adding 1M IPTG, incubated overnight• Cells were centrifuged

• Resuspended pellet was the crude sample of protein• Cells were lysed by sonification

• Resultant solution was the lysate same of protein

Protein Growth and Over-Expression of GFP-PuOx

Q-Sepharose Anion Exchange Column of GFP-PuOx

• The Q-sepharose is a positively charged resin

• GFP-PuOx is a negatively charged molecule, so it will be attracted to the Q-sepharose and stick to the column when added

• Using a KCl concentration gradient, salting out occurred on the column and PuOx was eluted out

• PuOx interacted with the positive potassium ion • The negative chloride ion interacted with the positive resin

• Experiment completed in the cold room

Socratic

GFP-PuOx After Purification• A280 completed on each of the

eluted fractions• Three peaks were found, each

peak distance was combined to create three separate samples

• Sample 1: tubes 4-11• Sample 2: tubes 12-18• Sample 3: tubes 19-24

• Samples were concentrated using 30,000 MWCO Amicon Filters

• About 250 L of each peak were 𝜇recovered

Activity Assay of GFP-PuOx• Amplex Red Assay used to check for

activity• The generation of H2O2 is coupled to

the conversion of the Amplex Red reagent to fluorescent resorufin by HRP5.

• Of the three fractions, only one showed any activity

• Activity shown by development of bright pink color

• This assay was used for all subsequent activity assays and kinetics assays

Thermo Fisher Scientific

Hydrogen Peroxide Standard Curve• A standard curve for hydrogen peroxide was built using

the Amplex Red Assay• Calculated the extinction coefficient to be 66, 615 M-1cm-1

• Literature value is 58,000 ± 5000 M-1cm-1

• This was later used to transform the kinetics data

SDS-PAGE of GFP-PuOx• Gel electrophoresis showed that the fractions

were not well purified using the Q-sepharose column

• Possible reasons• Q-sepharose not regenerated properly• Q-sepharose stock was overused • Salting out not adequate• Q-sepharose was not this optimal method of purification

16 kDa17 kDa

28 kDa

38 kDa

49 kDa

62 kDa98 kDa

188 kDa

6 kDa

Fractions

13 2

4 kDa

Histidine Tag Affinity Column of MBP-PuOx • Cell growth, overexpression, and lysis were

completed on cell stocks of MBP-PuOx as previously completed with GFP-PuOx

• Used 2X LB broth for overexpression• Nickel Column

• Histidine tag on PuOx has an affinity for the Nickel ion on the resin in the column• Imidazole has a higher affinity for Nickel ions than Histidine so will

knock off PuOx• Unbound proteins were washed from the column with a buffer containing a higher

concentration of Imidazole.• The concentration of Imidazole was increased again with elution

buffer to elute PuOx out of the column

Gentaur BVBA

FAD Cofactor Content• Spectral Scan from 300 nm - 600 nm completed on purified protein

sample to determine FAD content • The absorbance of the sample at 450 nm was used to find the ratio of FAD to PuOx

• Ratio of FAD to PuOx in the sample was 1:1.68 ration• Ratio also expressed at .5945 FAD/PuOx monomer• The expected ratio is 1:2

• The ratio was later used to transform the kinetics data

Enzyme Kinetics of MBP-PuOx

Enzyme Kinetics of MBP-PuOx

Kcat Kcat/Km

Experimental 13.78s-1 211.93 s-1mM-1

Literature 20.7s-1 5900 s-1mM-1

SDS-PAGE of MBP-PuOx• Gel electrophoresis showed a

heightening of purity through the stages of purification of the protein

• The stages of protein farther along the purification process had less bands than previous stages

• There was a noticeable jump of purification from the wash stage to the elutant stage

STD

Crude

Lysa

teLo

ading

Was

hEl

utan

t

16 kDa17 kDa

28 kDa

38 kDa

49 kDa

62 kDa

98 kDa

188 kDa

6 kDa

Conclusion• Purified MBP-PuOx• Determined Kinetics of purified MBP-PuOx• Determined FAD cofactor Content of MBP-PuOx• Learned and improved different laboratory protocols

Bibliography1. van Hellemond, E.W.; van Dijk, M.; Heuts, D.P.H.M.; Janssen, D.B; Fraaije, M.W. Discovery and

characterization of a putrescine oxidase from Rhodococcus erythropolis. Appl. Microbiol Biotechnol. (2008) 78:455-463.

2. Sanders, Jeremy K. M., and Sophie E. Jackson. "The Discovery and Development of the Green Fluorescent Protein, GFP." Chemical Society Reviews Chem. Soc. Rev. 38.10 (2009): 2821. Nobel Prize. Kungl Vetenskapsakademien The Royal Swedish Academy of Sciences, 8 Oct. 2008. Web. 2 Dec. 2015.

3. "Maltose Binding Protein Expression." New England Biolabs . N.p., n.d. Web. 2 Dec. 2015.4. "During an anion exchange chromatography experiment, 350 mM KCl in 15 mM Tris is added to elute

the protein. What is the exchange ion? Cl- , Tris-base, K+, or Tris." Socratic. N.p., 25 Feb. 2015. Web. 4 Dec. 2015.

5. Principle of coupled enzymatic assays using our Amplex® Red. Thermo Fisher Scientific, Waltham, MA. Web. 5 Dec. 2015.

6. Ni-IDA-Agarose. 2015. Gentaur BVBA, Belgium. Web. 4 Dec. 2015.